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Open Access Publications from the University of California

Specific communication between enhancers and alternative promoters mediates finer regulation of transcription initiation

  • Author(s): Bardales Mendieta, Jorge Adalberto
  • Advisor(s): Darzacq, Xavier
  • et al.
Abstract

Enhancers are DNA regulatory elements that play an important role in the precise

regulation of Pol II transcription initiation and, consequently, in the generation and

maintenance of patterns of gene expression. A defining characteristic of enhancers is their

capacity to regulate transcription from their target promoter, independent of the genomic

distance that separates them. Importantly, in order for enhancers to exert their regulatory

action, they must first relocalize into close spatial proximity to their target promoter and

form long-range interactions. Although these interactions have been demonstrated to

occur, the mechanisms that allow their specific and efficient formation remains largely

obscure. In this thesis, I focus on the development of tools and the expansion of our

understanding with regard to this important topic.

In the first chapter, I begin by providing a perspective of transcription initiation as

a hub of gene regulation. Then, I perform a concise overview of promoters and enhancers

as DNA regulatory elements and share the most important functional and structural

characteristics. I finalize the chapter by discussing the known regulatory mechanisms that

allow the formation of specific interactions between enhancers and promoters to take place.

In the second chapter, I present my findings on the development of imaging systems

to permit the visualization of enhancer-promoter interactions in live cells. I begin by

describing the limitations of current systems. Then, I discuss the importance of the

development of novel, innovative systems that allow specific labelling of genomic loci.

From there, I present the possibilities and limitations of a developed dCas9/Aptamer

system for the visualization of repetitive and non-repetitive loci. I also present my findings

on the development of a two-step integration system of operator arrays and share the

principal technical limitations encountered in its development. I finish by discussing and

providing a critical perspective of relevant published work that has been produced since

these efforts were undertaken.

In the third chapter, I begin by identifying and characterizing potential enhancers

in the PCSK9 locus in hepatic derived cells. I identify three candidate enhancers with

strong transcriptional activity by reporter assays. Then, I demonstrate how the regulatory

activity of these candidate enhancers is specific to hepatic cells and their regulatory activity

can be modulated by two small molecules in a similar fashion to the endogenous PCSK9

gene. From there, I dissect these DNA regulatory sequences to identify transcription factor

binding sites responsible for transcriptional activity by bioinformatic analysis and fine

mutagenic studies. These results point towards the presence of three DNA regulatory

elements required for PKCS9 expression.

In the fourth and final chapter, I investigate two alternative promoters (P1 and P2)

that drive MYC expression and are regulated by enhancers. The two MYC promoters can

be differentially regulated across cell-types, and their selective usage is largely mediated by

distal regulatory sequences. Moreover, in colon carcinoma cells, Wnt-responsive enhancers

prefer to upregulate transcription from the P1 promoter, using reporter assays in the

context of the endogenous Wnt induction. In addition, multiple enhancer deletions using

CRISPR/Cas9 corroborate the regulatory specificity of P1. Finally, I explore how

preferential activation between Wnt-responsive enhancers and the P1 promoter is

influenced by the distinct core promoter elements that are present in the MYC promoters.

Taken together, these results provide new insight into how enhancers can specifically target

alternative promoters and suggest that the presence of alternative promoters could create a

more efficient formation of long-range interactions and a more precise combinatorial

regulation of transcription initiation.

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